Human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV), both from the herpesvirus family, are among the most important opportunistic pathogens encountered in patients with AIDS. For example, CMV-associated complications, such as retinitis and gastroenteritis, are common in AIDS patients. KSHV-associated diseases, including Kaposi's sarcoma (KS) as well as primary effusion lymphoma (PEL), are among the leading neoplasms of AIDS patients. Moreover, HCMV and KSHV infections are also among the most common causes of oral diseases associated with AIDS patients (e.g. CMV-associated oral lesions and intraoral KS associated with KSHV). Blocking viral lytic replication systemically as well as locally in the infected tissues is central for the treatment of HCMV and KSHV systemic infections as well as viral-associated oral diseases. The goal of the proposed research is to develop small novel RNA-based molecules as antiviral therapeutic agents by directing endogenous RNase P to hydrolyze mRNAs coding for essential functions of HCMV and KSHV and to inhibit viral gene expression and replication. These RNA molecules, termed as external guide sequences (EGS), consist of a sequence antisense to the target mRNA and a sequence that guides RNase P to cleave the target. In this research program, we will first determine whether highly active and sequence-specific EGSs can be generated to direct human RNase P to cleave the mRNAs encoding the HCMV protease (PR) and KSHV immediate-early transcription activator, Rta. Moreover, we will use biochemical approaches to study the mechanism of how the generated EGSs efficiently direct RNase P to cleave the target mRNAs in vitro. Finally, we will determine whether these generated EGSs are highly effective in inhibiting HCMV and KSHV growth by abolishing the PR and Rta expression in cell culture. The proposed study will generate novel and exciting EGSs that can serve as lead compounds for anti-HCMV and anti-KSHV therapeutic development. Moreover, our study will provide insight into the mechanism of how EGSs achieve high targeting efficacy and sequence specificity and will generate guidelines for constructing effective EGSs for gene-targeting applications. These results will facilitate the development of these novel RNA-based agents for treatment and studies of AIDS-associated opportunistic infections, including those caused by HCMV and KSHV.